Insulin-like growth factors (IGFs) belong to a family of polypeptides known as somatomedins. At least two IGFs are known and are termed IGF-I and IGF-II, respectively. IGFs derive their name from the fact that they are structurally and functionally similar to insulin but are antigenically distinct from insulin.
IGF-I and IGF-II share a number of structural and biological properties. Both have molecular masses of about 7,500 daltons. IGF-I has 70 amino acid residues and IGF-II has 67 residues. Rinderknecht, J. Biol. Chem. (1978) 253:2769; and Rinderknecht, FEBS Lett. (1978) 89:283. IGF-I and IGF-II have 62% structural homology to each other. The molecules are single-chain polypeptides with three intrachain disulfide bridges. The IGFs include four peptide domains, A, B, C and D. The A and B domains are highly homologous to the corresponding domains of proinsulin and are linked by the C domain. The D domain exists as a carboxy terminal extension and a corresponding domain is not found in proinsulin. Like insulin, IGFs stimulate phosphorylation of specific tyrosine residues within the cytoplasmic domain of the receptors to which they bind. (See, e.g., WO 93/98826).
Both IGF-I and IGF-II have been isolated from human serum. The recombinant production of IGFs in bacterial and yeast hosts has also been described. For example, Chang and Swartz, Protein Folding: in vivo and in vitro (American Chemical Society, 1993) pp. 178–188, describe the recombinant production of IGF-I in E. coli. Elliott et al. J. Protein Chem. (1990) 9:95–104 describes the production of IGF-I in Saccharomyces cerevisiae using the α-factor pre-pro-leader to direct secretion of IGF-I into the culture medium. U.S. Pat. No. 5,324,639 describes the recombinant production of IGF-I in the methylotrophic yeast Pichia pastoris, using the S. cerevisiae alpha mating factor pre-pro sequence to direct secretion of the protein product.
However, attempts to purify authentic, properly folded IGF from recombinant hosts have been frustrated due to the tertiary structure of the molecule. In this regard, purification of the recombinantly produced molecule often renders a heterogenous mixture which consists largely of inactive, misfolded, insoluble and/or soluble disulfide-linked aggregates. Other aberrant molecules, such as fragments, nicked, oxidized and glycosylated forms, may also present. Thus, purification is difficult and yields of the authentic monomer are often low. See, e.g., Elliott et al. J. Protein Chem. (1990) 9:95–104.
Attempts have been made to correct these problems. For example, Chang and Swartz, Protein Folding: in vivo and in vitro (American Chemical Society, 1993) pp. 178–188, describe a method for solubilizing aggregated IGF-I produced in E. coli, using low concentrations of urea and dithiothreitol (DTT) in an alkaline buffer. U.S. Pat. No. 5,231,178, describes a method for the purification of correctly-folded, monomeric IGF-1 from P. pastoris using a combination of cation exchange, hydrophobic interaction, and gel filtration chromatography.
However, additional methods for the purification of authentic IGF from yeast would be desirable.